Chapter 5. Material and Methods
5.9. Bimolecular Fluorescence Complementation (BiFC)
region of LFY was cloned into pCL112 (pBATL) (construct made by Dr. Jennifer Pfluger) to create the nYFP. p35S:2xmCherry was cloned into pEarley102 (Earley et al., 2006) (construct made by Dr. Miin-Feng Wu). The control protein (NCb:TDY1-NLS in pCL113) was previously described (Ma et al., 2009). Constructs were transformed into onion epidermal cells using the PDS-1000/He Biolistic Particle Delivery System (BioRad, Hercules, CA, USA) as described by (Ma et al., 2009). Protein interactions were observed using an Olympus MVX10 fluorescent microscope.
Table 4. Genotyping primers
Sequence (5’ to 3’) Primer
Name Forward Reverse
LMI21 TCTGAAGGAGACCTGTAGTTGCTG GAGCCTGTGAGTAGCTGTCAATGT
lmi2-1 (LB1.3) ATTTTGCCGATTTCGGAAC GAGCCTGTGAGTAGCTGTCAATGT
lmi2-2 TCTGAAGGAGACCTGTAGTTGCTG (LB1.3) ATTTTGCCGATTTCGGAAC
LMI22 CTGAAAGCTTCACCAATCTCG AAATTCATTGCTTCCTTTGGC
lmi2-3 CTGAAAGCTTCACCAATCTCG (LB1.3) ATTTTGCCGATTTCGGAAC
lfy-13 AAGCAGCCGTCTGCGGTGTCAGCAG CTGTT CTGTCAATTTCCCAGCAAGACAC lfy-2 & lfy-104 AGAGAGACAGAGGGAGGATC TGTCGCATTTTAGGCTTGTTT AGL244 GAATGAGAGACATATTGGGAAGGT A AAGTGTCGGAGTCATCCTCAAG
agl24-3 (Lb1.3) ATTTTGCCGATTTCGGAAC GAGCCTGTGAGTAGCTGTCAATGT
ap1-105 CACATTTCTATCTAGGAAATCGATC
G
GTATGGCCTTCTCCTGTCATTTCC
ap1-116 TGAGCTTTTGGAGAGAAACCA AACGAAATAGCAGAAGGCAGTA
1
Gene specific primers for lmi2-1 and lmi2-2.2 Gene specific primers for lmi2-3. 3
Cut wild-type band with BstAP1. 4Cut wild-type band with BamH1.4 Gene specific primers for agl24-3. 5Cut with BS II. 6 Cut with Sca I.
Table 5. Cloning primers
Sequence (5’ to 3’)
Construct Forward Reverse
pLMI2:LMI2 aaaaagcaggctGAAACGTGTCTCCAC CCAAT agaaagctgggttCTAGAATTTGGAAACC ATGGA LMI2:GUS 1 GTCGACACTTGTAACTGTGCATG AAAC CCCGGGGATTGTTCCTCACCCCAC TAACA LMI2:GUS2 GAGCTCGTCTTATGAGAGCCTAA TATC CAATTGAATTTTCTCAAGCATTGT CAC
LMI2 in situ CTTTTATCTATGGGTCTTGATCCC GAATGGTTAATTGTTTAATGTTCT
GCAA
AP1 in situ CGGAATTCCTTACGCCGAAAGAC
AGCTT CGGGATCCCGTTCATTCTCTCTGA CCTTCA pLMI2:LMI2 -HA aaaaagcaggctGAAACGTGTCTCCAC CCAAT agaaagctgggttGAATTTGGAAACCATG GAAAC GST-LFY TAGAATTCATGGATCCTGAAGGT TTCAC ATGCGGCCGCCTAGAAACGCAAGT CGTCGC LMI2N GAGGTCGACCATGGGAAGAACAC CTTGTTG TTAGCGGCCGCCTATGATTCTCTA GAAAGCCTTGC LMI2C GAGGTCGACCAGAGAATCAATGC TCTTTAGC ATAGTTTAGCGGCCGCCTAGAATT TGGAAACCATGGA LFY3 aaaaagcaggctacATGGATCCTGAAGG TTTC agaaagctgggttCTAGAAACGCAAGTC GTC LFY4 CACCATGGATCCTGAAGGTTTCA CGAG GAAACGCAAGTCGTCGCC 2xmCherry4 CACCATGGTGAGCAAGGGCGAGG AG CTTGTACAGCTCGTCCATGCCG 35S:SOC1- GFP aaaaagcaggctacATGGTGAGGGGCAA AACTC agaaagctgggttCTTTCTTGAAGAACAA GGTAAC
Lower case sequences are attB1 and attB2 sequence specific. 1 Primers used to amplify
LMI2 upstream intergenic region. 2 Primers used to amplify LMI2 downstream intergenic region. 3 Primers used for yeast two hybrid constructs. 4 Primers used for BiFC constructs.
Table 6. Semi-quantitative and quantitative PCR (qRT-PCR) primers
Sequence (5’ to 3’)
Gene Forward Reverse
PCR cycle LMI21 (P1)TCATTGCTTCCTTTGGCT TT (P2) CTCATCTTCTTCAGGCGTCC 32 LMI21 (P3) GACGCCTGAAGAAGATG AGG (P4) TGTGGCGAGTTGTTGGTGA AGAT 32 EIF4A2 AAACTCAATGAAGTACTTGA GGGACA TCTCAAAACCATAAGCATAAAT ACCC 24 EIF4A 12 GCCATGGGTCTTCAAGAGAA CCCTTACAGAAGGGGACGAT NA AP12 GAAGGCCATACAGGAGCAA A ACTGCTCCTGTTGAGCCCTA NA AP12 AGGGAAAAAATTCTTAGGGC TCAACAG GCGGCGAAGCAGCCAAGGTTGC AGTTG NA AP3 GCCCTAACACCACAACGAAG G CTCACCTAGCCTCTGCTTGATC NA CAL CATTTCAACACCCCCATCTT GCCGTTTGGTCTTCTTCTTG NA FUL TTGCAAGATCACAACAATTC GCTTCT GAGAGTTTGGTTCCGTCAACGA CGAT NA LFY ACGCCGTCATTTGCTACTCT CTTTCTCCGTCTCTGCTGCT NA
LMI1 ATGGCCGGAGTCTAGTTCCT GTTGTTCGGAAATCGGTACG NA
LMI22 GACGCCTGAAGAAGATGAG
G
GAATGGCATGAAGCTGGATAA NA
LMI22 CGACCTCATCTGCACTTCTG CGCCACAGTAACCTCTTTCC NA
LMI22 GGGAAGAACACCTTGTTGTG
A
CCAGCTTCCATGTCCATTTT NA
LMI3 GAACGAATGGGACACGTTAT CAGACAATTCAGGATTGCCAG NA
LMI4 AATGGTGTCCGGTGAGATTT CATAACCACCGAATCCAACC NA
LMI5 AACTGGTGGTCGAACAGCTC AAGTGCATCTTCCCACATCC NA
AGL24 GAGGCTTTGGAGACAGAGTC GGTG AGATGGAAGCCCAAGCTTCAGG GA NA AG CAAAACTCCAACAGGCAATT G CATTTTCAGCTATCTTTGCAC NA 1
Primers used for T-DNA allele analysis. 2 Primer sets used interchangeably for quantitative PCR. Both sets give similar results. NA: not applicable-primers used for quantitative PCR.
Table 7. ChIP q-PCR primers
Sequence (5’ to 3’) Locus
(Region) Forward Reverse
AP1 (1) CAAAGCTTAATGGGCCTTGA GTCCGTGAGCTTTGTTTTGG
AP1 (2) TCGAACGTGGTGGTTAGAAG CGCAGCAGCTAGCATCTATTT
AP1 (3) AGAATGGTGGGGCTAAAAGC CAATCCAGCCACATCAAATG
AP1 (4) CAAACCTTCCTGCCTTCTTTT AATATCTCGATCCACTAAGATACG
G
AP1 (5) GCAAATGCCGAATCTGTTTT AAAAACCTTTGCTCAATTTGC
AP1 (6) ACACTTGGGGAAGGACCAGT ATGTCGGGTCCATGATTTTT
AP1 (7) GGGGGTCTTTGTTTTGTTTG CCCTTCCCATTTTTGATCCT
AP1 (8) AATGTGTCGCATCTAAGAAGATTT TCGAGTTCTAACTGCGGTTTC
AP1 (9) TGGGTTGTTAATGTTGATGTGTG TGGACTCGTACATAAGTTGGTTC
LFY (1) GCCAGTATTGCCAACTTTCC GGCCAACCTACGTCTTTTTC
LFY (2) TCACCACAGTGAAAACCCTAA TGTGTCTTGCTGGGAAATTG
LFY (3) GGAACCCAACGAGAGCATT TCTAAACCACCAAGTCGCATC
LFY (4) CTTTCGTTGGGAGCTTCTTG AGCGTGATGAGTACCGGAAT
LFY (5) ATGGGGTATGGTAGGGGAAC TGAAAACCCTGAGAAATCGTG
LFY (6) TTGATGTTGGGGAAAATGTG TCCTGATTTCTTCGCGTACC
LMI2 (1) TGAGACTCCCTTTGACTTGG AATTCCGTGGAAGCAAAAAT
LMI2 (2) ATGGACCCCACTGAGTGTCT TGCTGAGCTATTTACTTCAATTTCA
LMI2 (3) AGCTCGCTGATCGTCCTTT AAGCCAAAGGAAGCAATGAA
LMI2 (4) TTTCCGATAAGCGATGATGA CAGAAGACCCAAAAAGAAGCA
SVP (1) ACCTCACCAGTTGTGTCACG ACATCCATCAATTGGCGTTT
SVP (2) ATGATGATTGTGGCGATTGA TTCACCAACGTCAACAACAGA
SVP (3) GCCCTTGATGTTCTTCAGGT TGGGGAATTTCTTTTTATAGGG
SVP (4) GAGCCACCGACTAAGGTACG TGATCATGTAAACACACAGTTAGA
AA
EIF4 TGTTTTGCTTCGTTTCAAGGA GCATTTTCCCGATTACAAC
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